Periodic Reporting for period 1 - TITINmap (Towards Uncovering the Splicing Code of the Gigantic Gene Titin in Familial Dilated Cardiomyopathy)
Reporting period: 2022-01-01 to 2022-12-31
Titin is the largest protein of the human body and critical for the contraction of heart muscle cells. It is mutated in 30% of patients with familial dilated cardiomyopathy (DCM), a common heart disease that is a global threat to the aging society. RBM20, which is also mutated in patients with an aggressive form of DCM, regulates the length and function of TTN by a process named alternative splicing. How RBM20 mediates alternative splicing of TTN is largely unknown. I seek to develop a method to analyze RBM20-dependent splicing of TTN and other crucial targets in single cells. Using this tool, termed TITIN-seq, together with complementary stem cell-based assays, I seek to analyze changes in the repertoire of TTN isoforms upon disease-relevant mutations in the RBM20 gene. Moreover, TITIN-seq is used to identify novel splice regulators of TTN, which, together with RBM20, could complete the picture of alternative splicing of TTN. The overarching goal is to construct a comprehensive map of TTN splicing by integrating data of all its isoforms in single cells and its regulatory proteins. I envision that knowledge of such a splice map can be exploited for developing therapeutic strategies to revert aberrant TTN splicing in patients with DCM.
During my outgoing phase at Stanford, I have moved forward with several milestones regarding the TITINmap project. I have successfully generated and validated TTN splice reporter iPSCs using EXCISER tool as proposed (deliverable 1.5). Moreover, I evaluated several routes for cardiac differentiation in 2D and 3D and established a protocol for monitoring TTN isoform splicing (deliverable 1.1). Finally, I have successfully established TITIN-seq, a protocol enabling the monitoring of splice isoforms of TTN and other cardiac genes (deliverable 1.2/1.4). Combining TITIN seq and an optimized differentiation protocol for cardiomyocytes, I generated wild-type and RBM20 mutant cardiomyocytes and performed TITIN-seq to analyze the isoform distribution of RBM20-regulated genes in single cells (deliverable 2.1/2.2). Also I have already begun establishing a genetic screen for TTN splice regulators proposed in WP3.
Parts related to this project were summarized in a publication on Biorxiv which is currently in revision at Nature Communication.
Parts related to this project were summarized in a publication on Biorxiv which is currently in revision at Nature Communication.
During the second year, I expect to expand the single cell sequencing method TITIN-seq by adding CRISPR perturbations and their read-out by long-read sequencing. This would represent a novel technology to directly probe RNA binding proteins for their ability to regulate RNA splicing in a high-throughput fashion. So far, this is not possible as traditional high throughput single cell methods with perturbations only include the transcript`s ends and therefore a lot of information is lost.